Yh Samaranayake

Biofilm-Forming Ability of Candida albicans Is Unlikely To Contribute to High Levels of Oral Yeast Carriage in Cases of Human Immunodeficiency Virus Infection

ABSTRACT An increased prevalence of candidal carriage and oral candidiasis is common in cases of human immunodeficiency virus (HIV) infection, and the reasons for this may include the enhanced ability of colonizing yeasts to produce biofilms on mucosal surfaces. The aim of the present study was therefore to examine the differences, if any, in the biofilm-forming abilities of 26 Candida albicans yeast isolates from HIV-infected individuals and 20 isolates from HIV-free individuals, as this attribute of yeast isolates from patients with HIV disease has not been examined before. Biofilm formation in microtiter plate wells was quantitatively determined by both the 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino) carbonyl]-2H-tetrazolium hydroxide (XTT) reduction method and the crystal violet method. Although candidal biofilm formation could be quantitatively evaluated by either technique, the better reproducibility (P < 0.05) of the XTT reduction assay compared with that of the crystal violet method led us to conclude that the former is more reliable. There were no significant quantitative differences in biofilm formation between C. albicans isolates from HIV-infected patients and isolates from HIV-free individuals during in vitro incubation in a multiwell culture system over a period of 66 h. Three of eight host factors in the HIV-infected group were found to be associated with candidal biofilm formation. Thus, yeasts isolated from older individuals and those with higher CD4-cell counts exhibited decreased biofilm formation, while the findings for yeasts from individuals receiving zidovudine showed the reverse (P < 0.05 for all comparison). Our data indicate that attributes other than biofilm formation may contribute to the increased oral yeast carriage rates in cases of HIV infection.

Experimental Oral Candidiasis in Animal Models

SUMMARY Oral candidiasis is as much the final outcome of the vulnerability of the host as of the virulence of the invading organism. We review here the extensive literature on animal experiments mainly appertaining to the host predisposing factors that initiate and perpetuate these infections. The monkey, rat, and mouse are the choice models for investigating oral candidiasis, but comparisons between the same or different models appear difficult, because of variables such as the study design, the number of animals used, their diet, the differences in Candida strains, and the duration of the studies. These variables notwithstanding, the following could be concluded. (i) The primate model is ideal for investigating Candida-associated denture stomatitis since both erythematous and pseudomembranous lesions have been produced in monkeys with prosthetic plates; they are, however, expensive and difficult to obtain and maintain. (ii) The rat model (both Sprague-Dawley and Wistar) is well proven for observing chronic oral candidal colonization and infection, due to the ease of breeding and handling and their ready availability. (iii) Mice are similar, but in addition there are well characterized variants simulating immunologic and genetic abnormalities (e.g., athymic, euthymic, murine-acquired immune deficiency syndrome, and severe combined immunodeficient models) and hence are used for short-term studies relating the host immune response and oral candidiasis. Nonetheless, an ideal, relatively inexpensive model representative of the human oral environment in ecological and microbiological terms is yet to be described. Until such a model is developed, researchers should pay attention to standardization of the experimental protocols described here to obtain broadly comparable and meaningful data.

Candida krusei: biology, epidemiology, pathogenicity and clinical manifestations of an emerging pathogen

Early reports of Candida krusei in man describe the organism as a transient, infrequent isolate of minor clinical significance inhabiting the mucosal surfaces. More recently it has emerged as a notable pathogen with a spectrum of clinical manifestations such as fungaemia, endophthalmitis, arthritis and endocarditis, most of which usually occur in compromised patient groups in a nosocomial setting. The advent of human immunodeficiency virus infection and the widespread use of the newer triazole fluconazole to suppress fungal infections in these patients have contributed to a significant increase in C. krusei infection, particularly because of the high incidence of resistance of the yeast to this drug. Experimental studies have generally shown C. krusei to be less virulent than C. albicans in terms of its adherence to both epithelial and prosthetic surfaces, proteolytic potential and production of phospholipases. Furthermore, it would seem that C. krusei is significantly different from other medically important Candida spp. in its structural and metabolic features, and exhibits different behaviour patterns towards host defences, adding credence to the belief that it should be re-assigned taxonomically. An increased awareness of the pathogenic potential of this yeast coupled with the newer molecular biological approaches to its study may facilitate the continued exploration of the epidemiology and pathogenesis of C. krusei infections.

Antifungal effects of lysozyme and lactoferrin against genetically similar, sequential Candida albicans isolates from a human immunodeficiency virus-infected southern Chinese cohort.

ABSTRACT A variety of innate defense factors in saliva such as lysozyme and lactoferrin contribute to mucosal protection and modulateCandida populations in the oral cavity. It is also known that in human immunodeficiency virus (HIV)-infected individuals significant variations in the concentrations of lysozyme and lactoferrin in saliva occur during disease progression. Therefore, the aim of this study was to determine the in vitro susceptibility to human lactoferrin and hen egg white lysozyme of genotypically similar oralCandida albicans isolates obtained from six HIV-infected ethnic Chinese during sequential visits over a 12-month period. The similarity of the genotypes (50 in total) was evaluated using a randomly amplified polymorphic DNA assay. A blastospore viability assay was performed to evaluate the sensitivity of the organisms to lysozyme and lactoferrin. Exposure to physiological concentrations of either lysozyme (30 μg/ml) or lactoferrin (20 μg/ml) caused a rapid loss of viability among all isolates to a varying extent. None of the sequential C. albicans isolates demonstrated significant differences in sensitivity to either protein from one visit to the next; similar results were noted when the different genotypes from the same individual were compared. On Spearman correlation analysis of two genotypes that were sequentially isolated from a single patient, a significant negative correlation between lysozyme (r = −0.88; P < 0.02) (but not lactoferrin) resistance and the duration of HIV disease was seen. These results imply that a minority of C.albicans isolates that persist intraorally in individuals with HIV disease develop progressive resistance to innate salivary antifungal defenses such as lysozyme, possibly as an adaptive response. However, the vast majority of the Candidaisolates appear to succumb to these nonspecific host immune mediators abundantly present in the oral environment.

Architectural analysis, viability assessment and growth kinetics of Candida albicans and Candida glabrata biofilms

The human fungal pathogen Candida is able to form biofilms in almost all the medical devices in current use. Indeed, biofilm formation is a major virulence attribute of microorganisms and account for a majority of human infections. Therefore, understanding processes appertaining to biofilm development is an important prerequisite for devising new strategies to prevent or eradicate biofilm-related infections. In the present study we used an array of both conventional and novel analytical tools to obtain a comprehensive view of Candida biofilm development. Enumeration of colony forming units, colorimetric (XTT) assay, Scanning Electron Microscopy (SEM) and novel Confocal Laser Scanning Microscopy (CLSM) coupled with COMSTAT software analyses were utilised to evaluate growth kinetics; architecture and viability of biofilms of a reference (ATCC) and a clinical strain each of two Candida species, C. albicans and C. glabrata. Biofilm growth kinetics on a polystyrene substrate was evaluated from the initial adhesion step (1.5 h) up to 72 h. These analyses revealed substantial inter- and intra-species differences in temporal organisation of Candida biofilm architecture, spatiality and cellular viability, while reaching maturity within a period of 48 h, on a polystyrene substrate. There were substantial differences in the growth kinetics upon methodology, although general trend seemed to be the same. Detailed architectural analysis provided by COMSTAT software corroborated the SEM and CSLM views. These analyses may provide a strong foundation for down stream molecular work of fungal biofilms.

Cell Density and Cell Aging as Factors Modulating Antifungal Resistance of Candida albicans Biofilms

ABSTRACT Biofilm formation is a major virulence attribute of Candida pathogenicity which contributes to higher antifungal resistance. We investigated the roles of cell density and cellular aging on the relative antifungal susceptibility of planktonic, biofilm, and biofilm-derived planktonic modes of Candida. A reference and a wild-type strain of Candida albicans were used to evaluate the MICs of caspofungin (CAS), amphotericin B (AMB), nystatin (NYT), ketoconazole (KTC), and flucytosine (5FC). Standard, NCCLS, and European Committee on Antibiotic Susceptibility Testing methods were used for planktonic MIC determination. Candida biofilms were then developed on polystyrene wells, and MICs were determined with a standard 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino)carbonyl]-2H-tetrazolium hydroxide assay. Subsequently, antifungal susceptibility testing was performed for greater inoculum concentrations and 24- and 48-h-old cultures of planktonic Candida. Furthermore, Candida biofilm-derived planktonic cells (BDPC) were also subjected to antifungal susceptibility testing. The MICs for both C. albicans strains in the planktonic mode were low, although on increasing the inoculum concentration (up to 1 × 108 cells/ml), a variable MIC was noted. On the contrary, for Candida biofilms, the MICs of antifungals were 15- to >1,000-fold higher. Interestingly, the MICs for BDPC were lower and were similar to those for planktonic-mode cells, particularly those of CAS and AMB. Our data indicate that higher antifungal resistance of Candida biofilms is an intrinsic feature possibly related to the biofilm architecture rather than cellular density or cellular aging.

Relationship between the cell surface hydrophobicity and adherence of Candida krusei and Candida albicans to epithelial and denture acrylic surfaces

C. krusei is an emerging pathogen, especially in immunocompromised hosts, and is implicated, together with Candida albicans, as an etiological agent of oral candidoses. As the cell surface hydrophobicity of these yeasts appears to be important in the pathogenesis of superficial candidoses, 20 oral isolates of C. krusei and 5 oral isolates of C. albicans were investigated using a biphasic (hydrocarbon/aqueous) separation hydrophobicity assay. All the C. krusei isolates demonstrated significantly greater hydrophobicity than the C. albicans isolates (p<0.001). Further, there were significant intra‐species differences in cell surface hydrophobicity amongst C. krusei isolates. When hydrophobicity and adhesion to surfaces were compared using data from a previous study, there was a positive correlation between the cell surface hydrophobicity and adhesion of C. krusei isolates to HeLa surfaces (r=0.53, p<0.05), but not to acrylic surfaces. The current data, while confirming the inter‐ and intraspecies differences in cell surface hydrophobicity of Candida species, indicate that this attribute together with other cell surface features may determine the hierarchy of virulence amongst the different Candida species.

In Vitro Method To Study Antifungal Perfusion in Candida Biofilms

ABSTRACT Antimycotic perfusion through Candida biofilms was demonstrated by a modification of a simple in vitro diffusion cell bioassay system. Using this model, the perfusion of three commonly used antifungal agents, amphotericin B, fluconazole, and flucytosine, was investigated in biofilms of three different Candida species (i.e., Candida albicans, Candida parapsilosis, and Candida krusei) that were developed on microporous filters. Scanning electron microscopy revealed that C. albicans formed a contiguous biofilm with tightly packed blastospores and occasional hyphae compared with C. parapsilosis and C. krusei, which developed confluent biofilms displaying structural heterogeneity and a lesser cell density, after 48 h of incubation on nutrient agar. Minor structural changes were also perceptible on the superficial layers of the biofilm after antifungal perfusion. The transport of antifungals to the distal biofilm-substratum interface was most impeded by C. albicans biofilms in comparison to C. parapsilosis and C. krusei. Fluconazole and flucytosine demonstrated similar levels of perfusion, while amphotericin B was the least penetrant through all three biofilms, although the latter appeared to cause the most structural damage to the superficial cells of the biofilm compared with the other antifungals. These results suggest that the antifungal perfusion through biofilm mode of growth in Candida is dependent both on the antimycotic and the Candida species in question, and in clinical terms, these phenomena could contribute to the failure of Candida biofilm-associated infections. Finally, the in vitro model we have described should serve as a useful system to investigate the complex interactions that appear to operate in vivo within the biofilm-antifungal interphase.

Interspecies variation in Candida biofilm formation studied using the Calgary biofilm device.

An in vitro assay to study multiple Candida biofilms, in parallel, has been carried out using the Calgary biofilm device (CBD). We here report: i) standardization of the CBD for Candida albicans biofilm formation, ii) kinetics of C. albicans biofilm formation, iii) biofilm formation by five Candida species, and iv) effect of dietary carbohydrates on biofilm formation. The biofilm metabolic activity on all CBD pegs was similar (p=0.6693) and C. albicans biofilm formation revealed slow growth up to 36 h and significantly higher growth up to 48 h (p<0.001). Significant differences in total biofilm metabolic activity were seen for glucose, fructose and lactose grown C. albicans compared with sucrose and maltose grown yeasts. Candida krusei developed the largest biofilm mass (p<0.05) relative to C. albicans, C. glabrata, C. dubliniensis and C. tropicalis. Scanning electron microscopy revealed that C. krusei produced a thick multilayered biofilm of pseudohyphal forms embedded within the polymer matrix, whereas C. albicans, C. dubliniensis and C. tropicalis biofilms consisted of clusters or chains of cells with sparse extracellular matrix material. We conclude that CBD is a useful, simple, low cost miniature device for parallel study of Candida biofilms and factors modulating this phenomenon.

Candidiasis and other fungal diseases of the mouth

The advent of the human immunodeficiency virus (HIV) and the increasing prevalence of immunocompromised individuals due to surgical and medical advances have resulted in a resurgence of opportunistic infections including oral candidiasis and other rare mycoses which were once considered exotic. It is now recognized that oral candidiasis may present in many clinical guises that may confound the unwary clinician. Other mycotic diseases such aspergillosis, cryptococcosis, histoplasmosis, and mucormycosis may manifest intraorally both as primary lesions and as secondary manifestation of systemic disease. The primary oral pathology of most of the latter mycoses is ulcerations that respond well to systemic therapy with the polyene, amphotericin B. In general, the management of oral fungal infections has been revolutionized by the triazole group of drugs, fluconazole and itraconazole, although recent reports indicate an alarming increase of resistant organisms in particular to fluconazole. The first part of this review attempts to provide an overview of clinical variants of oral candidiasis and current therapeutic techniques, while the latter part outlines the rare oral mycoses and their management.